An important area of use of illuminating units is imaging units and examining units which are provided for the purpose of generating images of an object or a specimen to be examined. Typical examples for such imaging units are microscopes and especially microscopes having a wide field optic which images a pregiven region of the specimen, which is to be imaged or examined, and not only a small point-shaped region of the specimen, onto an image plane. The fluorescence examination has a special role for the optical examination of specimens. Here, the specimen is irradiated with excitation radiation having a suitable excitation spectrum which is selected in dependence upon one or several fluorescence colorants. If these fluorescence colorants are in the specimen, then they interact with the excitation radiation and emit fluorescence radiation which is characteristic for the fluorescence colorant. In this way, a detection of the fluorescence colorants in a specimen is possible. Not only can the presence of the fluorescence colorants be determined but also their concentration can be determined.
An important area of application of fluorescence examinations is molecular biology. Here, fluorescence colorants are used which specifically bond to pregiven substances in a biological specimen and can then be detected in the bonded state. A conclusion as to the presence and the concentration of the pregiven substance in the specimen is possible. For such examinations, so-called fluorescence readers are used which are designed for examining biochips having several regions (mostly even very many regions) delimited from each other with respectively different chemical compositions.
In lieu of fluorescence colorants, also nanoparticles, for example, quantum dots, can be used which fluoresce at at least one wavelength.
Two basic problems occur in such fluorescence examinations. First, the fluorescence intensities are often very low so that influences of other optical radiation in adjacent wavelength ranges or other origins on the detection of fluorescence radiation should be reduced as much as possible in order to obtain a favorable signal-to-noise ratio. For this purpose, illuminating devices are typically used which output excitation radiation with an only very narrow excitation spectrum. On the other hand, emission filters are placed in the detection beam path for the fluorescence radiation from the specimen to a detector. The emission filters preferably pass only radiation in the wavelength range of the fluorescence radiation.
Secondly, for a pregiven fluorescence colorant, the spectrum of the radiation with which the fluorescence of the colorant is excitable as well as also the fluorescence spectrum are specific so that different combinations of excitation radiation and emission filters are necessary for the detection of different fluorescence colorants.
Accordingly, it would be desirable for the rapid examination of specimens as to different fluorescence colorants to have an imaging and examining arrangement which permits a change between the different excitation spectra and/or emission spectra.
For this purpose, a filter wheel having different emission filters can, for example, be used in lieu of a fixed emission filter in the detector beam path. Each one of the emission filters is provided for a pregiven fluorescence spectrum. A white light source can especially be used as an illumination source. This solution has, inter alia, the disadvantage that, on the one hand, only a sequential examination of a specimen as to several fluorescence colorants can be carried out and, on the other hand, mechanical parts must be moved for a change between the examinations.
Additionally or alternatively, a multispectral illumination device can be used with which sequentially and/or parallelly optical radiation can be outputted in at least two different wavelength ranges. Optical radiation in at least two different wavelength ranges is understood to be radiation having an intensity with a pronounced maximum in the wavelength ranges. The intensity must not necessarily vanish in the region between the two wavelength ranges.
In United States patent application publication US 2006/0187542, a device for illuminating objects with light at different wavelengths is described for microscopes, automatic microscopes and apparatus for fluorescence microscopic applications. This device includes LED light sources for object illumination which are arranged in the illuminating beam path of the microscope or apparatus. For moving at least one of the luminescent diodes into an illuminating beam path, a mounting device, which is rotatable about a rotational axis, is provided and has holders for at least one of the luminescent diodes. The mounting device is adjustable via a drive unit so that the luminescent diode can be moved with the centroid wavelength into the illuminating beam path with the centroid wavelength being needed for the particular measurements and/or observations.
This solution too is disadvantageous in that mechanically movable parts are needed and only a sequential examination is possible.
The use of mechanically movable parts has several disadvantages. Thus, the change from the detection of one fluorescent colorant to another takes a certain time which delays the examination of specimens. Furthermore, the assembly is complicated and subjected to wear. Finally, it is very complex to satisfy the required accuracy conditions for the adjustment of the filters or the light sources to the detection beam path or illumination beam path.
In U.S. Pat. No. 6,372,485 and United States patent application publication US 2005/0224692 A1, illuminating devices having three LEDs are described. The radiation of these luminescent diodes is coupled into the same illuminating beam path. The described devices have, however, the disadvantage that the power of the luminescent radiation in the desired wavelength range is not very high compared to the radiation power of the luminescent diodes. Furthermore, the not so compact configuration does not allow a high light-conductance value. Furthermore, with three LEDs, no adequate spectral range for the excitation of fluorescence radiation can be covered.